The anterior pituitary gland contains at least five terminally differentiated cell types, which includes somatotropes, lactotropes, thyrotropes, corticotropes, and gonadotropes. Each of these cells has highly specialized endocrine functions to produce hormones that have diverse effects on mammalian physiology. Sparingly little information is currently known about the developmental requirements for pituitary-specific gene expression. To date, only one pituitary-specific gene transcription factor has been discovered, called Pit-1/GHF1. RNA transcripts encoding Pit-1 are found in all pituitary cells, but protein expression is found only in somatotropes, lactotropes and thyrotropes. Despite its ability to transactivate both growth hormone and prolactin promoters, the role of Pit- 1 in TSHbeta gene expression in thyrotropes in unclear. Specifically, it is unable to stimulate TSHbeta subunit promoter activity in both homologous thyrotrope cells and in cells of non-pituitary origin. I have now discovered another pituitary-specific transcription factor whose mRNA and protein expression are restricted to cells of thyrotropic origin,. This factor is a variant isoform of Pit-1 and specifically transactivates the thyrotrope-specific TSHbeta subunit gene. I have called this new factor Pit-1T, to emphasize its structural similarity to Pit-1 and its restricted expression in thyrotrope cells. The overall goal of this grant proposal is to gain specific information on the role of Pit-1 and Pit-1T in thyrotrope cells. To achieve these goals, I propose three specific aims. First, specific protein-DNA interactions between Pit-1T and the TSHbeta promoter will be studied. I will use DNase I protection analysis, mutational experiments with the TSHbeta promoter, and gel mobility shift analysis to better understand these interactions. Secondly, I will take advantage of the unique Pit-1T transactivation domain to further analyze the protein-protein interactions that mediate the TSHbeta promoter specific effect of Pit-1T. I will use Far Western blot analysis and subsequent Far Western library screening to identify these interactions. Finally, I will use the information generated from the first two specific aims to reconstitute TSHbeta promoter activity and gene expression in a thyrotrope-derived cell line which has lost the ability to express the TSHbeta gene. I will use stable transfections of Pit-1, Pit-1T, and other identified factors into the thyrotrope-derived alphaTSH cell line to achieve this goal. Acquisition of new knowledge gained from these studies will advance our understanding of TSHbeta gene expression in thyrotropes, and provide insight into the complex molecular mechanisms governing cell-specific regulation of highly restricted genes.